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Abstract:

Methods for laser microdissection isolation of viable cells are provided.
Cells of a desired type may be isolated from a diverse population,
optionally with detection and exclusion of undesired cells. Desired cells
may be isolated from a population that arose from differentiation of
pluripotent cells, preferably embryonic stem cells or induced pluripotent
stem cells, and undifferentiated stem cells may be detected and excluded
from selection including the isolation of RPE cells sleeted based on
morphology (e.g., characteristic mottled appearance) from a population of
ES cells. The cells isolated by these methods, including RPE cells, may
be essentially free of undifferentiated cells and thus suitable for use
in cell-based therapies.

Claims:

1-66. (canceled)

67. A method for isolating a viable cell from a heterogeneous population
of cells comprising (a) providing a planar carrier on which said
population of cells containing said at least one viable cell is situated,
(b) placing said culture dish in a microscope coupled to a laser
microdissection system, (a) selecting said viable cell, (b) excising said
viable cell, (c) separating said viable cell from the planar carrier, and
(d) collecting said viable cell.

68-78. (canceled)

79. The method of claim 67, wherein said viable cell is produced by
culturing pigmented epithelial cells obtained from differentiated
embryonic stem cells.

80. The method of claim 67, wherein said viable cell is an RPE cell
selected based on pigmentation.

81. The method of claim 67, wherein said viable cell is an RPE cell
selected based on at least one detectable characteristic of RPE cells.

82. The method of claim 81, wherein said detectable characteristic of RPE
cells includes at least one of presence of brown pigmentation accumulated
within the cytoplasm, a cobblestone, epithelial-like morphology, or
expression of at least one RPE cell markers.

83. The method of claim 82, wherein said RPE cell marker is selected from
the group consisting of bestrophin, RPE65, CRALBP, and PEDF.

84. The method of claim 83, wherein said marker is detected by a method
selected from the group consisting of binding to an antibody directly or
indirectly coupled to a detectable label; incubation with magnetic
beads-conjugated antibodies; detecting the expression of a fluorescent
protein; detecting an intracellular mRNA, detecting an intracellular
protein; and detecting an intracellular small molecule.

85. (canceled)

86. The method of claim 67, wherein excising of step (d) comprises
removing the selected cells from the planar carrier using
micromanipulation or laser catapulting.

102. A method for isolating a RPE cell from a population of cells
comprising (a) providing a planar carrier on which said population of
cells is situated, (b) placing said planar carrier in a microscope
coupled to a laser microdissection system, (c) selecting said at least
one RPE cell, (d) excising said cell from undesired cells or other
materials in target areas adjacent to the selected cells using laser
light, thereby severing the connections between the selected cells and
adjacent cells or other materials, and (e) collecting said RPE cell.

104. The method of claim 102, wherein said population of cells is a
heterogeneous population.

105. The method of claim 102, wherein said RPE cell is differentiated
from one or more pluripotent cells.

106-138. (canceled)

139. A method of isolating a viable RPE cell from a heterogeneous
population of cells comprising (a) providing a planar carrier on which a
cell population comprising at least one viable desired cell is situated;
(b) selecting at least one desired cell to be isolated; (c) excising said
at least one cell from undesired cells or other materials in target areas
adjacent to the selected cells using laser light, thereby severing the
connections between the selected cells and adjacent cells or other
materials; and (d) separating the at least one selected cell from the
planar carrier, thereby isolating the selected cells, wherein the
isolated cells comprise viable desired cells, wherein said desired cells
are of a desired cell type selected from the group consisting of iris
pigment epithelium cells, vision-associated neural cells, lens cells, rod
cells, cone cells, or corneal cells.

140. The method of claim 139, wherein said RPE cell is differentiated
from one or more pluripotent cells.

141. (canceled)

142. (canceled)

143. The method of claim 139, wherein said selected cell exhibits at
least one detectable characteristics of RPE cells.

144. The method of claim 143, wherein said detectable characteristics of
RPE cells includes morphology or expression of at least one RPE cell
markers.

[0004] Laser microdissection methods may allow for the isolation of an
individual cell to be separated from the surrounding preparation (e.g., a
tissue section) by the laser beam, and then released. The released cells
may then be moved to a collection device, for example by mechanical means
or a laser-induced transport process with the aid of a laser pulse. See
Thalhammer, et al. (2003) Laser Methods in Medicine and Biology 13(5):
681-691 and Murray & Curran (2005) Laser Microdissection: Methods and
Protocols 293 from Methods in Molecular Biology.

[0005] Laser-mediated micromanipulation (LMM), a laser microdissection
technique, uses a fine, focused laser beam to sever the connections
between desired cells and the surrounding portion of the specimen. The
desired cells may then be removed by physical manipulation or by
"catapulting" (e.g., a laser pulse imparts momentum to the desired piece
and allows to be moved without being touched). See Thalhammer, et al.
(2003) Laser Methods in Medicine and Biology 13(5): 681-691.

[0007] In an another laser microdissection technique, laser capture
microdissection (LCM), a thermoplastic film is placed over the sample and
caused to selectively adhere to the desired cells by a laser pulse that
heats part of the thermoplastic film and causes it to adhere to the
cells. The cells are then removed with the film. Cells isolated by LCM
have been used for analysis of DNA, RNA, and protein. See, e.g., Buck, et
al. (1996) Science 274(5289): 998-1001, Bonner, et al. (1997) Science
278(5342): 1481-1483; U.S. Pat. No. 6,184,973; U.S. Pat. No. 6,897,038;
U.S. Pat. No. 5,859,699; U.S. Pat. No. 6,495,195; U.S. Pat. No.
6,100,051; U.S. Pat. No. 6,720,191; U.S. Pat. No. 6,700,653; and U.S.
Pat. No. 6,743,601.

[0008] The LCM technique has been used to isolate cells for extraction and
analysis of their contents. For example, ethanol-fixed cells have been
isolated by LCM from post-mortem human eyes for RT-PCR measurement of
alterations in gene expression in retinal pigment epithelium cells
adjacent to basal deposits. Yamada, et al. (2006) Exp Eye Res. 82(5):
840-8. Similar techniques--again using post-mortem human eyes,
ethanol-fixation, and RT-PCR analysis--have been used to identify
differences in gene expression between retinal pigment epithelium cells
isolated by LCM from the different regions of the eye. Ishibashi, et al.
(2004) Invest Ophthalmol Vis Sci. 45(9): 3291-301. Retinal pigment
epithelium and other cells have also been isolated by LCM from frozen
mouse eye sections for RT-PCR to determine which specific cell type(s)
expressed cytokines in inflamed eyes. Foxman, et al. (2002) J Immunol.
168(5): 2483-92. These references report using LCM to isolate non-viable
cells for molecular analysis but do not report using LCM to isolate
viable cells.

[0009] Laser microdissection and pressure catapulting (LMPC), a laser
microdissection technique, involves placing a biological sample directly
on top of a thermoplastic polyethyelene napthalate membrane that covers
the glass slide. The membrane acts as a support (scaffolding) to allow
for catapulting relatively large amounts of intact material at a time. A
focused laser beam is used to cut out an area of the membrane and
corresponding biological sample, and the beam is then defocused and the
energy used to catapult the membrane and material from the slide. The
catapulted sample may be captured in an aqueous media positioned directly
above the cut area. See Kuhn, et al. (2007) Am J Phyiol. Heart Circ.
Physiol. 292: H1245-H1253, H1245. This method has been used to isolate
embryonic-stem cells derived cardiomyocytes. Khuram, et al. (2006)
Toxicological Sciences 90(1): 149-158, abstract.

[0010] However, there remains a need for improved techniques for isolating
ocular cells (e.g., retinal pigment epithelium cells, iris pigment
epithelium cells, vision-associated neural cells, lens cells, rods,
cones, and corneal cells) that remain viable and which are of sufficient
purity as to be useful for cell-based therapies.

[0013] Degeneration of the RPE may cause retinal detachment, retinal
dysplasia, or retinal atrophy that is associated with a number of
vision-altering ailments that result in photoreceptor damage and
blindness, such as, choroideremia, diabetic retinopathy, macular
degeneration (including age-related macular degeneration), retinitis
pigmentosa, and Stargardt's Disease (fundus flavimaculatus). See WO
2009/051671.

RPE Cells in Medicine

[0014] Given the importance of the RPE in maintaining visual and retinal
health, the RPE and methodologies for producing RPE cells in vitro are of
considerable interest. See Lund, et al. (2001) Progress in Retinal and
Eye Research 20(4): 415-449. For example, a study reported in Gouras, et
al. (2002) Investigative Ophthalmology & Visual Science 43(10): 3307-311
describes the transplantation of RPE cells from normal mice into
transgenic RPE65.sup.-/- mice (a mouse model of retinal degeneration).
Gouras discloses that the transplantation of healthy RPE cells slowed the
retinal degeneration in the RPE65.sup.-/- mice but after 3.7 weeks, its
salubrious effect began to diminish. Treumer, et al. (2007) Br J
Opthalmol 91: 349-353 describes the successfully transplantation of
autologous RPE-choroid sheet after removal of a subfoveal choroidal
neovascularization (CNV) in patients with age related macular
degeneration (AMD), but this procedure only resulted in a moderate
increase in mean visual acuity.

[0015] However, RPE cells sourced from human donors has several
intractable problems. First, is the shortage of eye donors, and the
current need is beyond what could be met by donated eye tissue. For
example, RPE cells sourced from human donors are an inherently limited
pool of available tissue that prevent it from scaling up for widespread
use. Second, the RPE cells from human donors may be contaminated with
pathogens and may have genetic defects. Third, donated RPE cells are
derived from cadavers. The cadaver-sourced RPE cells have an additional
problem of age where the RPE cells are may be close to senesce (e.g.,
shorter telomeres) and thus have a limited useful lifespan following
transplantation. Reliance on RPE cells derived from fetal tissue does not
solve this problem because these cells have shown a very low
proliferative potential. Further, fetal cells vary widely from batch to
batch and must be characterized for safety before transplantation. See,
e.g., Irina Klimanskaya (2009) Retinal Pigment Epithelium Derived From
Embryonic Stem Cells, in STEM CELL ANTHOLOGY 335-346. Any human sourced
tissue may also have problems with tissue compatibility leading to
immunological response (graft-rejection). Also, cadaver-sourced RPE cells
may not be of sufficient quality as to be useful in transplantation
(e.g., the cells may not be stable or functional). Fourth, sourcing RPE
cells from human donors may incur donor consent problems and must pass
regulatory obstacles, complicating the harvesting and use of RPE cells
for therapy. Fifth, a fundamental limitation is that the RPE cells
transplanted in an autologous transplantation carry the same genetic
information that may have lead to the development of AMD. See, e.g.,
Binder, et al. (2007) Progress in Retinal and Eye Research 26(5):
516-554. Sixth, the RPE cells used in autologous transplantation are
already cells that are close to senesce, as AMD may develop in older
patients. Thus, a shorter useful lifespan of the RPE cells limits their
utility in therapeutic applications (e.g., the RPE cells may not
transplant well and are less likely to last long enough for more complete
recovery of vision). Seventh, to be successful in long-term therapies,
the transplanted RPE cells must integrate into the RPE layer and
communicate with the choroid and photoreceptors. Eighth, in AMD patients
and elderly patients also suffer from degeneration of the Bruch's
membrane, complicating RPE cell transplantation. See Gullapalli, et al.
(2005) Exp Eye Res. 80(2): 235-48. Thus there exists a great need for a
source of RPE cells for therapeutic uses and human embryonic stem cells
(hES) are considered a promising source of replacement RPE cells for
clinical use. See Idelson, et al. (2009) Cell Stem Cell 5: 396-408.

[0016] Methods for the systematic directed matter for the production of
large numbers of RPE cells have been described (e.g., PCT/US2010/57056
and WO 2009/051671). For example, when differentiated cells are to be
produced from ES cells for transplantation, there is concern that
presence of a few residual ES cells could give rise to a tumor or
teratoma. Some assurance of safety can come from administering the cell
preparation to an animal (e.g., an immune compromised animal). However,
animal testing alone may be considered insufficient because a human ES
cell may be more prone to produce a teratoma in a human host than in the
animal model.

[0017] Additionally, methods for producing RPE cells by differentiation of
RPE cells from pluripotent stem cells produces a heterogeneous population
of cells comprising RPE cells and other differentiated cells (e.g.,
neural rosettes). The standard method of manual selection relies on the
operator's skill and experience in selecting the RPE cells and not the
other differentiated cells. Moreover, manual selection of pigmented
clusters is very tedious and fully relies on the operator's skills and
judgment which may get impaired after several hours of such scrupulous
selection and the microscope involving eye and back-straining work. Thus,
it is desirable to provide methods that may decrease or eliminate the
possibility of undesired residual undifferentiated ES cells in a cell
population isolated from differentiated ES cells. Thus, there exists a
need for a rapid method for the isolation of large numbers of RPE cells
with sufficient purity as to be suitable for use in transplantation
therapies.

BRIEF SUMMARY OF THE INVENTION

[0018] In one aspect, the invention provides a method for isolating a
viable cell morphologically distinguishable from other cells contained
within a heterogeneous population of cells comprising (a) providing a
planar carrier containing a heterogeneous cell population, (b) placing
said planar carrier in a microscope coupled to a laser microdissection
system, (c) selecting said desired cell, (d) excising said cell, and (e)
collecting said cell.

[0019] In one embodiment, the population of cells may comprise human or
primate cells. In another embodiment, the population may comprises both
differentiated and undifferentiated cells. The undifferentiated cells may
comprise embryonic stem cells (ESCs). The embryonic stem cells may be
identified by detection of a detectable characteristic selected from the
group consisting of presence in a round colony with clear margins; a high
nucleus/cytoplasm ratio with prominent nucleoli; rounded cells that lie
tightly packed with each other; and expression of at least one ES cell
markers selected from the group consisting of OCT-4, Nanog, TRA-1-60,
SSEA-3, SSEA-4, TRA-1-81, SOX2, and alkaline phosphatase.

[0020] In one embodiment, the cell population may be produced by
differentiation of embryonic stem cells. In another embodiment, the
differentiation of embryonic stem cells may comprise (a) allowing hES
cell cultures to overgrow on MEF and form a thick multilayer of cells, or
forming an embryoid body (EB) from hES cells; (b) culturing the hES cells
multilayer of cells or EB for a sufficient time for the appearance of
pigmented cells comprising brown pigment dispersed in their cytoplasm.

[0021] In one embodiment, the cell may be produced by culturing pigmented
epithelial cells obtained from differentiated embryonic stem cells. In a
further embodiment, the method may further comprise contacting said cell
of step (a) with a vital stain. In another embodiment, the excising of
step (d) may comprise removing the selected cells from the planar carrier
using micromanipulation or laser catapulting. In a further embodiment,
the collection of step (e) may comprise manual colony picking,
micromanipulation, or laser capture.

[0022] In one aspect, the invention provides a method for isolating a
viable differentiated cell which is morphologically distinguishable from
other undifferentiated cells which both are contained a population of
cells comprising (a) providing a planar carrier on which said population
of cells containing said at least one differentiated cell is situated,
(b) placing said planar carrier in a microscope coupled to a laser
microdissection system, (c) selecting said differentiated cell, (d)
excising said differentiated cell, (e) separating said differentiated
cell from the planar carrier, and (f) collecting said differentiated
cell.

[0023] In one embodiment, the population of cells may be a heterogeneous
population. population comprises both differentiated and undifferentiated
cells. In another embodiment, the undifferentiated cells comprise
embryonic stem cells (ESCs). In another embodiment, the embryonic stem
cells may be identified by detection of a detectable characteristic
selected from the group consisting of presence in a round colony with
clear margins; a high nucleus/cytoplasm ratio with prominent nucleoli;
rounded cells that lie tightly packed with each other; and expression of
at least one ES cell markers selected from the group consisting of OCT-4,
Nanog, TRA-1-60, SSEA-3, SSEA-4, TRA-1-81, SOX2, and alkaline
phosphatase.

[0025] In one embodiment, the pluripotent stem cell may be an embryonic
stem cell. In another embodiment, the differentiation of embryonic stem
cells may comprise (a) allowing hES cell cultures to overgrow on MEF and
form a thick multilayer of cells, or forming an embryoid body (EB) from
hES cells; (b) culturing the hES cells multilayer of cells or EB for a
sufficient time for the appearance of pigmented cells comprising brown
pigment dispersed in their cytoplasm. In another embodiment, the
differentiated cell may be produced by culturing pigmented epithelial
cells obtained from differentiated embryonic stem cells.

[0026] In another embodiment, the method may further comprise contacting
said cell of step (a) with a vital stain. In another embodiment, the
excising of step (d) may comprise removing the selected cells from the
planar carrier using micromanipulation or laser catapulting. In another
embodiment, the collection of step (e) may comprise manual colony
picking, micromanipulation, or laser capture.

[0027] In one aspect, the invention provides a method for isolating a
viable cell from a heterogeneous population of cells comprising (a)
providing a planar carrier on which said population of cells containing
said at least one viable cell is situated, (b) placing said culture dish
in a microscope coupled to a laser microdissection system, (c) selecting
said viable cell, (d) excising said viable cell, (e) separating said
viable cell from the planar carrier, and (f) collecting said viable cell.

[0028] In one embodiment, the population may comprise both differentiated
and undifferentiated cells. In another embodiment, the undifferentiated
cells may be pluripotent stem cells.

[0029] In one embodiment, the pluripotent stem cell may be an embryonic
stem cell (ESC). In another embodiment, the embryonic stem cells may be
identified by detection of a detectable characteristic selected from the
group consisting of presence in a round colony with clear margins; a high
nucleus/cytoplasm ratio with prominent nucleoli; rounded cells that lie
tightly packed with each other; and expression of at least one ES cell
markers selected from the group consisting of OCT-4, Nanog, TRA-1-60,
SSEA-3, SSEA-4, TRA-1-81, SOX2, and alkaline phosphatase.

[0030] In one embodiment, the cell population is produced by
differentiation of embryonic stem cells. In another embodiment, the
differentiation of embryonic stem cells may comprise (a) allowing hES
cell cultures to overgrow on MEF and form a thick multilayer of cells, or
forming an embryoid body (EB) from hES cells; (b) culturing the hES cells
multilayer of cells or EB for a sufficient time for the appearance of
pigmented cells comprising brown pigment dispersed in their cytoplasm.

[0031] In one embodiment, the viable cell may be produced by culturing
pigmented epithelial cells obtained from differentiated embryonic stem
cells. In another embodiment, the method may further comprise contacting
said cell of step (a) with a vital stain. In another embodiment, the
excising of step (d) may comprise removing the selected cells from the
planar carrier using micromanipulation or laser catapulting. In another
embodiment, the collection of step (e) may comprise manual colony
picking, micromanipulation, or laser capture.

[0032] In one aspect, the invention provides a method for isolating a RPE
cell from a population of cells comprising (a) providing a planar carrier
on which said population of cells is situated, (b) placing said planar
carrier in a microscope coupled to a laser microdissection system, (c)
selecting said at least one RPE cell, (d) excising said cell from
undesired cells or other materials in target areas adjacent to the
selected cells using laser light, thereby severing the connections
between the selected cells and adjacent cells or other materials, and (e)
collecting said RPE cell.

[0033] In one embodiment, the population of cells may be a heterogeneous
population. In another embodiment, the population may comprise both
differentiated and undifferentiated cells.

[0034] In one embodiment, the undifferentiated cells may comprise
embryonic stem cells (ESCs). In another embodiment, the embryonic stem
cells may be identified by detection of a detectable characteristic
selected from the group consisting of presence in a round colony with
clear margins; a high nucleus/cytoplasm ratio with prominent nucleoli;
rounded cells that lie tightly packed with each other; and expression of
at least one ES cell markers selected from the group consisting of OCT-4,
Nanog, TRA-1-60, SSEA-3, SSEA-4, TRA-1-81, SOX2, and alkaline
phosphatase.

[0035] In one embodiment, the cell population may be produced by
differentiation of embryonic stem cells. In another embodiment, the
differentiation of embryonic stem cells may comprise (a) allowing hES
cell cultures to overgrow on MEF and form a thick multilayer of cells, or
forming an embryoid body (EB) from hES cells; (b) culturing the hES cells
multilayer of cells or EB for a sufficient time for the appearance of
pigmented cells comprising brown pigment dispersed in their cytoplasm.

[0036] In one embodiment, the RPE cell is produced by culturing pigmented
epithelial cells obtained from differentiated embryonic stem cells. In
another embodiment, the method may further comprise contacting said cell
of step (a) with a vital stain. In another embodiment, the excising of
step (d) may comprise removing the selected cells from the planar carrier
using micromanipulation or laser catapulting. In another embodiment, the
collection of step (e) may comprise manual colony picking,
micromanipulation, or laser capture.

[0037] In one aspect, the invention provides a method of isolating a
viable RPE cell from a heterogeneous population of cells comprising (a)
providing a planar carrier on which a cell population comprising at least
one viable desired cell is situated; (b) selecting at least one desired
cell to be isolated; (c) excising said at least one cell from undesired
cells or other materials in target areas adjacent to the selected cells
using laser light, thereby severing the connections between the selected
cells and adjacent cells or other materials; and (d) separating the at
least one selected cell from the planar carrier, thereby isolating the
selected cells, wherein the isolated cells comprise viable desired cells,
wherein said desired cells are of a desired cell type selected from the
group consisting of iris pigment epithelium cells, vision-associated
neural cells, lens cells, rod cells, cone cells, or corneal cells.

[0038] In one embodiment, the heterogeneous population may comprise both
differentiated and undifferentiated cells. In another embodiment, the
undifferentiated cells may comprise embryonic stem cells (ESCs). In
another embodiment, the embryonic stem cells may be identified by
detection of a detectable characteristic selected from the group
consisting of presence in a round colony with clear margins; a high
nucleus/cytoplasm ratio with prominent nucleoli; rounded cells that lie
tightly packed with each other; and expression of at least one ES cell
markers selected from the group consisting of OCT-4, Nanog, TRA-1-60,
SSEA-3, SSEA-4, TRA-1-81, SOX2, and alkaline phosphatase.

[0039] In one embodiment, the heterogeneous cell population may be
produced by differentiation of embryonic stem cells. In another
embodiment, the differentiation of embryonic stem cells may comprise (a)
allowing hES cell cultures to overgrow on MEF and form a thick multilayer
of cells, or forming an embryoid body (EB) from hES cells; (b) culturing
the hES cells multilayer of cells or EB for a sufficient time for the
appearance of pigmented cells comprising brown pigment dispersed in their
cytoplasm.

[0040] In one embodiment, the RPE cell may be produced by culturing
pigmented epithelial cells obtained from differentiated embryonic stem
cells. In another embodiment, the method may further comprise contacting
said cell of step (a) with a vital stain. In another embodiment, the
excising of step (d) may comprise removing the selected cells from the
planar carrier using micromanipulation or laser catapulting. In another
embodiment, the collection of step (e) may comprise manual colony
picking, micromanipulation, or laser capture.

[0041] In one embodiment, the viable cell may be a RPE cell selected based
on pigmentation. In another embodiment, the viable cell may be an RPE
cell selected based on at least one detectable characteristic of RPE
cells. The detectable characteristic of RPE cells may be at least one of
presence of brown pigmentation accumulated within the cytoplasm, a
cobblestone, epithelial-like morphology, or expression of at least one
RPE cell markers. The RPE cell marker may be selected from the group
consisting of bestrophin, RPE65, CRALBP, and PEDF. The RPE marker may be
detected by a method selected from the group consisting of binding to an
antibody directly or indirectly coupled to a detectable label; incubation
with magnetic beads--conjugated antibodies; detecting the expression of a
fluorescent protein; detecting an intracellular mRNA, detecting an
intracellular protein; and detecting an intracellular small molecule. The
viable cell may exhibit at least one detectable characteristics of RPE
cells. The detectable characteristics of RPE cells may be morphology or
expression of at least one RPE cell markers. The RPE cell marker may be
selected from the group consisting of markers identified in Table 1.

[0042] In one embodiment, the differentiated cell may be a RPE cell
selected based on pigmentation. In another embodiment, the differentiated
cell may be an RPE cell selected based on at least one detectable
characteristic of RPE cells. The detectable characteristic of RPE cells
may be at least one of presence of brown pigmentation accumulated within
the cytoplasm, a cobblestone, epithelial-like morphology, or expression
of at least one RPE cell markers. The RPE cell marker may be selected
from the group consisting of bestrophin, RPE65, CRALBP, and PEDF. The RPE
marker may be detected by a method selected from the group consisting of
binding to an antibody directly or indirectly coupled to a detectable
label; incubation with magnetic beads-conjugated antibodies; detecting
the expression of a fluorescent protein; detecting an intracellular mRNA,
detecting an intracellular protein; and detecting an intracellular small
molecule. The differentiated cell may exhibit at least one detectable
characteristics of RPE cells. The detectable characteristics of RPE cells
may be morphology or expression of at least one RPE cell markers. The RPE
cell marker may be selected from the group consisting of markers
identified in Table 1.

[0043] In one embodiment, the viable cell may be differentiated from one
or more pluripotent cells. In another embodiment, the pluripotent cells
may be selected from the group consisting of induced pluripotent stem
(iPS) cells, embryonic stem (ES) cells, blastomeres, morula cells,
embroid bodies, adult stem cells, hematopoietic stem cells, fetal stem
cells, mesenchymal stem cells, postpartum stem cells, multipotent stem
cells, and embryonic germ cells. In a further embodiment, the pluripotent
stem cell may be an embryonic stem cell. In a still further embodiment,
the pluripotent stem cell may be a human embryonic stem cell.

[0044] In one embodiment, the differentiated cell may be differentiated
from one or more pluripotent cells. In another embodiment, the
pluripotent cells may be selected from the group consisting of induced
pluripotent stem (iPS) cells, embryonic stem (ES) cells, blastomeres,
morula cells, embroid bodies, adult stem cells, hematopoietic stem cells,
fetal stem cells, mesenchymal stem cells, postpartum stem cells,
multipotent stem cells, and embryonic germ cells. In a further
embodiment, the pluripotent stem cell may be an embryonic stem cell. In a
still further embodiment, the pluripotent stem cell may be a human
embryonic stem cell.

[0045] In one embodiment, the viable cell may be a differentiated cell. In
one embodiment, the differentiated cell may be a RPE cell. In a further
embodiment, the viable cell may be a RPE cell. In another embodiment, the
RPE cell may be a retinal pigment epithelium (RPE) cell. In another
embodiment, the RPE cell may be selected from the group consisting of
iris pigment epithelium cells, vision-associated neural cells, lens
cells, rod cells, cone cells, or corneal cells. In another embodiment,
the differentiated cell may be selected from the group consisting of iris
pigment epithelium cells, vision-associated neural cells, lens cells, rod
cells, cone cells, or corneal cells. In another embodiment, the viable
cell may be selected from the group consisting of iris pigment epithelium
cells, vision-associated neural cells, lens cells, rod cells, cone cells,
or corneal cells.

[0046] In a one embodiment, the viable cell may be a human viable cell. In
another embodiment, the viable cell may be a non-human animal, non-human
primate, murine, ovine, bovine, canine, porcine, chimpanzee, cynomolgus
monkey, baboon, Old World monkey, caprine, equine, ungulate, or feline
viable cell. In a one embodiment, the differentiated cell may be a human
viable cell. In another embodiment, the differentiated cell may be a
non-human animal, non-human primate, murine, ovine, bovine, canine,
porcine, chimpanzee, cynomolgus monkey, baboon, Old World monkey,
caprine, equine, ungulate, or feline differentiated cell. In a one
embodiment, the RPE cell may be a human viable cell. In another
embodiment, the viable cell may be a non-human animal, non-human primate,
murine, ovine, bovine, canine, porcine, chimpanzee, cynomolgus monkey,
baboon, Old World monkey, caprine, equine, ungulate, or feline RPE cell.

[0047] In one embodiment, the RPE cell may be differentiated from one or
more pluripotent cells. In another embodiment, the pluripotent cells may
be selected from the group consisting of induced pluripotent stem (iPS)
cells, embryonic stem (ES) cells, blastomeres, morula cells, embroid
bodies, adult stem cells, hematopoietic stem cells, fetal stem cells,
mesenchymal stem cells, postpartum stem cells, multipotent stem cells,
and embryonic germ cells. In a further embodiment, the pluripotent stem
cell may be an embryonic stem cell. In a still further embodiment, the
pluripotent stem cell may be a human embryonic stem cell.

[0048] In one embodiment, the collected cells may comprise differentiated
cells. In another embodiment, the collected cells may comprise RPE cells.
In another embodiment, the collected cells may consist of RPE cells. In a
further embodiment, the collected cells may comprise differentiated cells
and essentially no undifferentiated cells. In another embodiment, the
collected cells may comprise RPE cells and essentially no other
differentiated cells. In yet another embodiment, the collected cells may
comprise RPE cells and essentially no undifferentiated cells. In another
embodiment, the collected cells may comprise RPE cells and no pluripotent
stem cells. In still another embodiment, the collected cells may comprise
RPE cells and essentially no embryonic stem cells. In one embodiment, the
collected cells comprise viable cells. In another embodiment, the
collected cells consist of viable cells. In a further embodiment, the
collected cells do not comprise any undifferentiated cells.

[0054] In another embodiment, the laser light may be produced from a laser
selected from the group consisting of argon ion lasers, diode lasers, dye
lasers, excimer lasers, fiber lasers, free electron lasers, krypton ion
lasers, Nd: YAG lasers, Nd: YVO4 lasers, and solid-state bulk
lasers. In another embodiment, the laser light may be ultraviolet light.
In another embodiment, the laser light may be provided as pulses having a
duration between about 100 μs and about 3000 μs. In a further
embodiment, the laser light may be produced from the STILETTO® laser
system.

[0055] In one embodiment, the methods described herein may be conducted
under sterile conditions. In another embodiment, the method may further
comprise further comprising culturing the isolated viable cell.

[0056] In an embodiment, the method may further comprise at least one
additional round of laser isolation, each additional round of laser
isolation comprising isolating said cell from a cell population resulting
from the preceding round of laser isolation by the method according to
any one of the preceding claims.

[0057] In another embodiment, the method may further comprise at least one
additional rounds of laser isolation, each additional round of laser
isolation comprising isolating desired cells from a cell population
resulting from the preceding round of laser isolation by the method
according to any one of the preceding claims.

[0058] In a yet a still further embodiment, the invention provides a
purified population of RPE cells produced by a method described herein.

[0059] In a still further embodiment, the invention provides a method of
preventing or treating a disease of the retina comprising providing RPE
cells produced by the method of the forgoing claims; and introducing said
RPE cells into the eye of an affected individual. In one embodiment, the
disease of the retina may be selected from the group consisting of
retinal detachment, retinal dysplasia, retinal atrophy, choroideremia,
diabetic retinopathy, macular degeneration, age-related macular
degeneration, retinitis pigmentosa, and Stargardt's Disease (fundus
flavimaculatus). In another embodiment, the cells may be provided in a
suspension, matrix, or scaffold.

[0067] The invention relates to methods for isolation of viable cells
using laser microdissection. In particular, the laser microdissection
methods described herein may be used to isolate desired cells from a
diverse starting population (e.g., a mixed population of cells
differentiated from embryonic stem (ES) cells.) The invention provides
methods comprising laser microdissection that may produce a substantially
pure population of isolated cells (e.g., populations with few or no
undesired cell types present). The laser microdissection methods may
produce a substantially pure population of isolated cells which may be
more pure than populations produced by manual colony picking or chemical
separation methods (e.g., collagenase treatment). The substantially pure
populations may be suitable for cell transplantation or other therapeutic
uses because they contain few or no undesired cells. Surprisingly, it has
been found laser microdissection may be used to isolate a pure population
of desired cells from a heterogeneous population (e.g., differentiated
cells purified from a heterogeneous population including
non-differentiated and differentiated cells).

DEFINITIONS

[0068] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as those commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described herein can
be used in the invention or testing of the present invention, suitable
methods and materials are described below. The materials, methods and
examples are illustrative only, and are not intended to be limiting.

[0069] As used in the description herein and throughout the claims that
follow, the meaning of "a," "an," and "the" includes plural reference
unless the context clearly dictates otherwise.

[0070] "Embryo" or "embryonic," as used herein refers broadly to a
developing cell mass that has not implanted into the uterine membrane of
a maternal host. An "embryonic cell" may be a cell isolated from or
contained in an embryo. This also includes blastomeres, obtained as early
as the two-cell stage, and aggregated blastomeres.

[0071] "Embryonic stem cells" (ES cells), as used herein, refers broadly
to cells derived from the inner cell mass of blastocysts or morulae that
have been serially passaged as cell lines. The ES cells may be derived
from fertilization of an egg cell with sperm or DNA, nuclear transfer,
parthenogenesis, or by means to generate ES cells with homozygosity in
the HLA region. ES cells may also refer to cells derived from a zygote,
blastomeres, or blastocyst-staged mammalian embryo produced by the fusion
of a sperm and egg cell, nuclear transfer, parthenogenesis, or the
reprogramming of chromatin and subsequent incorporation of the
reprogrammed chromatin into a plasma membrane to produce a cell.
Embryonic stem cells, regardless of their source or the particular method
used to produce them, may be identified based on the: (i) ability to
differentiate into cells of all three germ layers, (ii) expression of at
least Oct-4 and alkaline phosphatase, and (iii) ability to produce
teratomas when transplanted into immunocompromised animals.

[0072] "Embryo-derived cells" (EDC), as used herein, refers broadly to
morula-derived cells, blastocyst-derived cells including those of the
inner cell mass, embryonic shield, or epiblast, or other pluripotent stem
cells of the early embryo, including primitive endoderm, ectoderm, and
mesoderm and their derivatives. "EDC" also including blastomeres and cell
masses from aggregated single blastomeres or embryos from varying stages
of development, but excludes human embryonic stem cells that have been
passaged as cell lines.

[0073] "Isolated," as used herein, describes cells that are substantially
free of at least one protein, molecule, or other impurity that is found
in its natural environment (e.g., "substantially purified".) The term
"isolated" may be used interchangeably with "purified."

[0074] "Laser microdissection system," as used herein, refers broadly to
any method using a laser to isolate cells from a sample, including but
not limited to laser capture microdissection (LCM), laser microdissection
and pressure catapulting (LMPC), laser microdissection (LMD), and
laser-assisted microdissection (LMD or LAM).

[0075] "Mature RPE cell" and "mature differentiated RPE cell," as used
herein, may be used interchangeably throughout to refer broadly to
changes that occur following initial differentiating of RPE cells.
Specifically, although RPE cells may be recognized, in part, based on
initial appearance of pigment, after differentiation mature RPE cells may
be recognized based on enhanced pigmentation.

[0076] "Multipotent cell," as used herein refers broadly to any cell that
has the potential to give rise to cells from multiple lineages within a
cell type (e.g., a hematopoietic cell--a blood cell that can develop into
several types of blood cells).

[0077] "Pigmented," as used herein refers broadly to any level of
pigmentation, for example, the pigmentation that initial occurs when RPE
cells differentiate from ES cells. Pigmentation may vary with cell
density and the maturity of the differentiated RPE cells. The
pigmentation of a RPE cell may be the same as an average RPE cell after
terminal differentiation of the RPE cell. The pigmentation of a RPE cell
may be more pigmented than the average RPE cell after terminal
differentiation of the RPE cell. The pigmentation of a RPE cell may be
less pigmented than the average RPE cell after terminal differentiation.

[0078] "Pluripotent stem cell," as used herein, refers broadly to a cell
capable of prolonged or virtually indefinite proliferation in vitro while
retaining their undifferentiated state, exhibiting normal karyotype
(e.g., chromosomes), and having the capacity to differentiate into all
three germ layers (i.e., ectoderm, mesoderm and endoderm) under the
appropriate conditions.

[0080] "RPE cell," "differentiated RPE cell," "ES-derived RPE cell," and
as used herein, may be used interchangeably throughout to refer broadly
to an RPE cell differentiated from a pluripotent stem cell using a method
of the invention. The term is used generically to refer to differentiated
RPE cells, regardless of the level of maturity of the cells, and thus may
encompass RPE cells of various levels of maturity. RPE cells may be
visually recognized by their cobblestone morphology and the initial
appearance of pigment. RPE cells may also be identified molecularly based
on substantial lack of expression of embryonic stem cell markers such as
Oct-4 and NANOG, as well as based on the expression of RPE markers such
as RPE-65, PEDF, CRALBP, and bestrophin. Thus, unless otherwise
specified, RPE cells, as used herein, refers to RPE cells differentiated
in vitro from pluripotent stem cells.

[0082] Generally, a laser may be coupled to a microscope and focused onto
the heterogeneous cell population (e.g., tissue) in a culture dish. By
movement of the laser by optics or the stage the focus follows a
trajectory which may be predefined by the user. This trajectory, the
element, may then be cut out and separated from the adjacent cells (e.g.,
tissue.) After the cutting process, a collection process may be used to
remove the target cells from the sample.

[0083] The laser microdissection systems may employ a variety of lasers
including but not limited to UV lasers (e.g., UV-A laser (˜355
nm)). Further, various computer systems for laser dissection are known in
the art and may be used in the methods described herein. For example, the
Stiletto® laser dissection system from Hamilton Thorne, Olympus
SmartCut® laser microdissection system, CellCut® laser
microdissection system with MMI CapLift®, or AutoPix® laser
capture microdissection system, ArcturusXT® laser capture
microdissection system may be used. Additionally, any one or all of the
steps of the methods described herein may be automated. Further, any one
or all of the steps of the methods described herein may be conducted
under sterile conditions.

[0084] In one aspect, the invention provides a method for isolating
differentiated cells from a heterogeneous population of cells comprising
placing a culture dish containing said heterogeneous cell population on a
microscope coupled to a laser dissection system, selecting differentiated
cells for isolation, excising the differentiated cells, and collecting
said differentiated cells.

[0085] In one aspect, the disclosure provides a method of isolating viable
cells comprising providing a planar carrier, placing a heterogeneous cell
population comprising differentiated cells; selecting at least one cells
to be isolated; excising the cells, thereby severing the connections
between the selected cells and adjacent cells or other materials; and
separating the selected cells from the planar carrier, thereby isolating
the selected cells. The desired cells are preferably ocular cells, and
most preferably RPE. The laser ablating may be automated, for example,
the user selects the cells to be isolated and provides information to a
computer running a laser cutting program (e.g., mmi SmartCut Plus, mmi
CellCut®). A high precision, motorized XY-stage may be controlled
through computer mouse or keyboard. The program may comprise an overview
that allows for navigation within the culture dish to facilitate
selection of the desired cells. Several positions of the stage may be
stored for returning to an area of interest. The program may comprise a
drawing tool where for marking the cutting path, the user may choose
between free hand drawing and geometric figures such as circles, squares
and ellipses for selecting desired cells. This allows the user to mark
objects over the entire slide area and these objects will the selected
area may be cut automatically by the computer. The size of the circles,
squares and ellipses may be chosen by the user and be copied via use of a
computer. In one embodiment, automation of the methods described herein
allows for the isolation of large amounts of highly pure populations RPE
cells differentiated from ES cells under sterile conditions in a reduced
period of time (compared to manual or chemical selection of RPE cells).
For example, the laser isolation method described herein may be fully
automated to allow for the isolation of RPE cells without manual or
chemical selection of RPE cells. This allows for significant savings in
cost (including labor) and time (e.g., isolated the cells in a matter of
hours instead of days or weeks).

[0086] In another aspect, laser microdissection and pressure catapulting
(LMPC) may be used. In LMPC, a heterogeneous population of cells in a
culture dish may be placed directly on top of a thermoplastic
polyethyelene napthalate membrane that covers the culture dish. The
membrane acts as a support (scaffolding) to allow for catapulting
relatively large amounts of intact material at a time. A focused laser
beam may be used to cut out an area of the membrane and corresponding
biological sample, and the beam may be then defocused and the energy used
to catapult the membrane and material from the slide. A motorized robotic
(e.g., RoboMover) stage may be used to move the sample through the laser
beam path to allow the user to control the size and shape of the area to
be cut. The catapulted sample may be captured in an aqueous media
positioned directly above the cut area. See Kuhn, et al. (2007) Am J
Phyiol. Heart Circ. Physiol. 292: H1245-H1253, H1245.

[0087] The starting population of cells may be differentiated from any
pluripotent cells. For example, the pluripotent cells may be embryonic
stem cells, induced pluripotent stem (iPS) cells, single blastomeres,
morula cells, embroid bodies, adult stem cells, hematopoietic cells,
fetal stem cells, mesenchymal stem cells, postpartum stem cells,
multipotent stem cells, or embryonic germ cells. In another embodiment,
the pluripotent stem cells may be mammalian pluripotent stem cells. In
still another embodiment, the pluripotent stem cells may be human
pluripotent stem cells including but not limited to human embryonic stem
(hES) cells, human induced pluripotent stem (iPS) cells, human adult stem
cells, human hematopoietic stem cells, human fetal stem cells, human
mesenchymal stem cells, human postpartum stem cells, human multipotent
stem cells, or human embryonic germ cells. In another embodiment, the
pluripotent stem cells may be a hES cell line listed in the European
Human Embryonic Stem Cell Registry--hESCreg. Also, the pluripotent stem
cells may be human embryonic stem cells (hES cells), human induced
pluripotent stem (iPS) cells, or embryonic stem cells of another species.
Further, the pluripotent stem cells of (a) may be genetically engineered.
The starting population of cells may comprise an embryoid body. For
example, a pluripotent stem cell may be differentiated to produce a
heterogeneous population comprising at least one differentiated cell. The
differentiated cell may then be isolated using laser microdissection
methods described herein. Further, the isolated cell may be further
cultured to expand the isolated population or to confirm the purity of
the isolated cells (e.g., culture the isolated cell to confirm the
absence of undesired cells).

[0088] The population of differentiated cells may be produced by culturing
ES cells using the methods disclosed in U.S. Pat. Nos. 7,795,025;
7,794,704; 7,736,896; U.S. patent application Ser. No. 12/682,712,
International Patent Application No. PCT/US2010/57056, and WO
2009/051671. For example, ES cells may be cultured as a multilayer
population or embryoid body for a sufficient duration for the appearance
of pigmented epithelial cells or other differentiated cell types, which
may then be isolated and further cultured. After differentiation, the ES
cell population produces a heterogeneous population of cells comprising
both undifferentiated ES cells and differentiated cells (e.g., RPE
cells). The differentiated cells may be distinguished from the
undifferentiated ES cells and other differentiated cells (e.g., non-RPE
cells) in the heterogeneous cell population based on color,
characteristic shape, size, cellular markers, or cellular functions
(e.g., enzymatic markers). For example, in a heterogeneous population of
cells comprising ES cells and RPE cells, the RPE cells are selected for
isolation based on morphological characteristics including but not
limited to pigmentation, a characteristic cobblestone, epithelial
appearance (mottled appearance), or RPE cells markers. The methods
described herein may comprise differentiating RPE cells from a cell
population of ES cells. The differentiated RPE cells may form
tightly-packed pigmented colonies. These colonies may be selected for
isolation using the laser microdissection methods described herein. In
one embodiment, the selection area may be totally within the pigmented
differentiated RPE cell colony. In this embodiment, no undifferentiated
ES cells are excised, yielding a pure population of differentiated RPE
cells (e.g., no ES cells). The selection area may be defined by the
boundary between the pigmented RPE cells and the undifferentiated ES
cells. See, e.g., FIGS. 1 and 3. A nearly pure population of
differentiated RPE cells may be isolated (e.g., essentially no ES cells
or other differentiated cells). These methods may also produce RPE cells
that are suitable for therapeutic use, such as treatment of macular
degeneration by cell transplantation into an affected eye.

[0089] Selection of cells for laser microdissection may be generally based
on detectable characteristics (e.g., presence of a cell marker, absence
of a cell marker, uptake of a dye, morphology, pigmentation). The cells
selected for isolation may exhibit at least one detectable
characteristics indicative of a desired cell, and/or may not exhibit at
least one detectable characteristics whose presence would indicate an
undesired cell. For example, when differentiated cells are to be isolated
from a population that arose by differentiation of ES cells, cells may be
selected for isolation only if they do not include any cells exhibiting
detectable characteristic(s) of ES cells.

[0090] At the time of their excision, the selected cells may be at least a
minimum specified distance from any undesired cells. For example, the
only cells in contact with the selected cells may be cells that exhibit
detectable characteristics indicative of desired cells and/or that do not
exhibit a detectable characteristic indicative of an undesired cell. As
another example, the selected cells may be fully contained within an
island of cells that exhibit the detectable characteristics being used to
identify desired cells, and/or adjacent to cell-free spaces. The minimum
specified distance may be specified in distance units (e.g., at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 micrometers), as a
number of cell widths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20
cell widths), and/or as a minimum layers of surrounding cells (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 cells) between the selected cells
and any cells that exhibit characteristics of undesired cells or that do
not exhibit the characteristics being used to identify desired cells.
Moreover, the distance may be at least about 1-2 μm. Such methods may
provide further assurance that the isolated cells are free from undesired
cells, e.g., due to the possibility that an individual undesired cell may
be more difficult to detect within a group of desired cells, and such an
undesired cell may be more likely to be located near the periphery of an
island of desired cells than internally.

[0091] Optionally, laser microdissection may be utilized for multiple
iterations, wherein cells are isolated by laser microdissection and
optionally cultured, and the resulting cell population may be subjected
to laser microdissection. Use of multiple iterations may provide even
greater assurance that undesired cells are not present, and may also help
ensure phenotypic stability and uniformity in the resulting population of
cultured cells. The cell(s) isolated during each round may also be
selected on the basis of a trait (e.g., level of expression of a
particular gene) to facilitate isolation of a more desirable cell
population. Cells may be isolated based on the same trait or different
traits during successive iterations.

[0092] The cells to be isolated are typically provided on a planar
carrier. The planar carrier used may be of any type, so long as it allows
light to pass through. Typically the support may be optically clear. In a
preferred embodiment the support may be polystyrene which may be
optionally tissue-culture treated polystyrene. Other suitable supports
may include glass (e.g., a glass slide or cover slip), polyethylene
terephthalate, polycarbonate, polyethylene, polypropylene Particularly
preferred carriers include microtiter plates (e.g., 6-, 12-24-, 96-,
384-, and 1536-well plates)

[0093] The cutting out of a target area may be preferably performed under
microscopic view. Alternatively, or in addition, the target area may be
visualized through use of an image recording device, such as a CCD
camera, which may be used to generate an image of the material located on
the carrier, and display it on a display device. This image may be
superimposed with a user interface of the laser microdissection system to
facilitate selecting the objects to be processed with the laser beam.

[0094] Preferably, the planar carrier may be movable within the
microscopic view, thereby facilitating isolation of desired cells from
various portions of the planar carrier. For example, the planar carrier
may be affixed to a moveable stage (e.g., an X-Y or X-Y-Z stage).
Movement of the planar carrier may be performed manually or may be
automated, for example, driven by a computer-controlled stepper motor.
For example, automated movement of the planar carrier during laser
ablation may be used to move the target areas into the laser beam path.
Exemplary moveable stages are available from Prior Scientific (e.g., the
PROSCAN® product lines).

[0095] The laser light may be typically focused to a small diameter and
applied to the sample, preferably from the bottom side of the support,
along a target position on the biological preparation, thereby cutting
out the biological preparation.

[0096] The laser light may be of any wavelength that may be used to excise
cells or other materials in target areas adjacent to the selected cells
while preferably retaining the viability of adjacent non-irradiated
cells. In a preferred embodiment, the laser light may be ultraviolet
light, e.g., having a wavelength less than about 400 nm. Preferably, the
wavelength may be between 200 and 400 nm, such as near-UV (between 400
and 300 nm), middle-UV (between 300 and 200 nm), UVA (between 400 and 320
nm), UVB (between 320 and 280 nm) or UVC (between 280 and 200 nm). Known
ultraviolet lasers and methods of producing ultraviolet laser light may
be utilized, including argon ion lasers; diode lasers (e.g., based on
gallium nitride); dye lasers; excimer lasers (including F2, ArF,
KrF, XeBr, or XeCl, XeF); fiber lasers such as neodymium-doped fluoride
fiber lasers; free electron lasers; krypton ion lasers; lasers producing
wavelengths longer than ultraviolet and incorporating non-linear
frequency conversion (such as an Nd: YAG or Nd: YVO4 laser coupled
to two successive frequency doublers); and solid-state bulk lasers
including cerium-doped crystals such as Ce3+: LiCAF or Ce3+:
LiLuF4 (which may optionally be pumped with nanosecond pulses from a
frequency-quadrupled Q-switched laser). Further exemplary laser systems
are described in U.S. Pat. Nos. 4,641,912; 4,773,414; 4,784,135;
4,785,806; 5,144,630; 5,146,465; 5,237,576; 5,742,626; 5,745,284; and
7,277,220.

[0097] The laser light may be delivered in pulses or continuously. For
example, the laser pulse length may be between 100 μs and 3000 μs,
or shorter or longer pulse durations may also be utilized. The duration
and frequency of laser pulses may be adjusted appropriately in such a
manner that a required amount of energy may be directed to a target area
to be cut. Preferably, the laser pulse duration and frequency are
sufficient to sever connections between the selected cells and
surrounding material, while retaining viability of the cells to be
isolated.

[0098] In a preferred embodiment, the laser module may be combined into a
single unit with an objective (e.g., a 20× objective), for example,
as a single compact turret mounted unit. A particularly preferred laser
module may be the STILETTO® laser system available from Hamilton
Thorne Ltd. (Beverly, Mass.).

[0099] The method may be performed manually or may include use of an
automated system. An automated system may perform at least one or all of
the steps of the method without the need for human intervention or with
human supervision or intervention. For example, based on the presence of
detectable characteristics an automated system may suggest cells for
isolation and/or suggest target areas for laser ablation, and a human
operator may accept, modify, or reject the suggestions by the automated
system.

[0100] Several ways for collecting cells which have been isolated from a
heterogenous population on a microscope slide (e.g., culture dish) are
known in the art. For example, the isolated cells may be collected by
pipette, washing, or laser pressure catapult.

[0101] For example, the excised cells may be catapulted by a photonic
cloud into a microcentrifuge tube cap. The cells may be attached to a cap
lined with a thermoplastic film that forms a protrusion when hit with a
laser pulse. The protrusion closes the gap between the cells and the
film. Lifting the cap may remove the target cells and keep them attached
to the cap. The cap may be then placed in a microcentrifuge tube for
processing. This cap method may be used in conjunction with cutting cells
from a tissue section and then attaching them to a cap. The cells may be
propelled using an electrostatic force toward a film, and then the film
may be pushed inside a microcentrifuge tube for collection.

[0102] In a cell ablation method, live cells in a sterile culture dish may
be covered with a light absorbing film. The laser may cut around the
cells of interest under the film and, when the film may be removed, the
cells stay in the culture dish and the unwanted cells (e.g.,
undifferentiated cells) come off with the film. This method is referred
to as "cell ablation" because it removes the unwanted cells from the
culture and the remaining cells may be washed and re-cultured. See
Bancroft & Gamble (2008) Theory and Practice of Histological Techniques,
page 575.

[0103] In a laser catapult method, the sample may be catapulted from a
culture dish by a defocused U.V laser pulse that generates a photonic
force propelling the material off the dish. This is also referred to as
Laser Micro-dissection Pressure Catapulting (LMPC) and the cells may be
sent upward (e.g., up to several mm) to a collection vessel (e.g.,
microfuge tube cap) containing buffer or a specialized material in the
tube cap that the cells may adhere to. This active catapulting process
avoids some of the static problems when using membrane-coated slides.
See, e.g., Zeiss PALM MicroBeam; U.S. Pat. Nos. 5,689,109; 5,998,129; and
6,930,714. Another similar LCM process cuts the sample from above and the
sample drops via gravity into a capture device below the sample. See
Leica Microsystems Laser Microdissection System. Further, the excised
cells may be collected by pipetting, or manual picking of the excised
cells after they are excised from the heterogeneous population in the
laser microdissection system.

[0104] Further, the methods described herein may be conducted under
sterile conditions. For example, the methods described herein may be
conducted in accordance with Good Manufacturing Practices (GMP) (e.g.,
the cultures are GMP-compliant) and/or current Good Tissue Practices
(GTP) (e.g., the cultures may be GTP-compliant.)

Isolated Cell Populations

[0105] The present invention provides purified preparations of desired
cells, preferably differentiated cells isolated from a heterogeneous
population comprising differentiated and non-differentiated cells (e.g.,
RPE cells isolated from a heterogeneous population of RPE cells, ES
cells, and differentiated cells). The desired cells isolated by the
methods described herein may be substantially free of at least one
protein, molecule, or other impurity that is found in its natural
environment (e.g., "isolated".) For example, the methods described herein
may provide isolated RPE cells, substantially purified populations of RPE
cells, and pharmaceutical preparations comprising RPE cells.

[0107] The desired cells isolated by the laser microdissection methods
described herein from a heterogeneous cell population that may comprises
a desired differentiated cell, differentiated cells that may not be
desired, and undifferentiated cells.

[0113] The differentiated cell cultures may be prepared in accordance with
Good Manufacturing Practices (GMP) (e.g., the cultures are GMP-compliant)
and/or current Good Tissue Practices (GTP) (e.g., the cultures may be
GTP-compliant.)

Retinal Pigment Epithelium (RPE) Cells

[0114] The present invention provides RPE cells that may be isolated from
a heterogeneous population of cells comprising, for example, pluripotent
cells, such as human embryonic stem cells or human iPSC's, and are
molecularly distinct from embryonic stem cells, adult-derived RPE cells,
and fetal-derived RPE cells. See, also, Liao, et al. (2010) Human
Molecular Genetics 19(21): 4229-4238. The inventors surprisingly
discovered that the method by which the RPE cells are isolated from a
heterogeneous population of cells, for example, pluripotent stem cells
from which they may be differentiated, may an important factor in
determining the purity of the resulting RPE cells. The inventors found
that the RPE cells produced by the methods described produced a
substantially pure RPE cell population (e.g., essentially no non-RPE
cells) than previous methods of isolated RPE cells. Further, the methods
described herein are less labor-intensive and faster than methods using
chemical agents (e.g., collagenase) or labor-intensive methods (e.g.,
manual colony picking). See, e.g., FIGS. 4 and 6, respectively. For
example, the isolation methods described herein allow for the rapid and
repeatable final RPE cell product of substantial purity (e.g.,
essentially no non-RPE cells). Further, the methods of isolating RPE
cells described herein that avoid the inclusion of ES cells in the final
RPE cell population. Thus, as ES cells are not present in any amount in
populations isolated by the methods described herein, and they do not
pose an unacceptable risk of contamination in the RPE cell cultures and
preparations.

[0115] The cell types that may be isolated from a heterogeneous cell
population by this invention include, but are not limited to, RPE cells,
RPE progenitor cells, iris pigmented epithelial (IPE) cells, and other
vision associated neural cells, such as internuncial neurons (e.g.,
"relay" neurons of the inner nuclear layer (INL)) and amacrine cells. The
invention also provides methods of isolating retinal cells, rods, cones,
and corneal cells as well as cells providing the vasculature of the eye
from heterogeneous population. Further, the methods described herein may
be used to isolated RPE cells from a heterogeneous population comprising
RPE cells, pluripotent stem cells, and other non-RPE differentiated
cells.

[0116] The RPE cells isolated by the methods described herein may be used
for treating retinal degeneration diseases due to retinal detachment,
retinal dysplasia, or retinal atrophy or associated with a number of
vision-altering ailments that result in photoreceptor damage and
blindness, such as, choroideremia, diabetic retinopathy, macular
degeneration (e.g., age-related macular degeneration), retinitis
pigmentosa, and Stargardt's Disease (fundus flavimaculatus).

[0117] The RPE cells may express at least one RPE cell marker that may be
used to identify the RPE cells in a heterogenous population for
isolation. For example, the RPE cells may express RPE65, PAX2, PAX6,
tyrosinase, bestrophin, PEDF, CRALBP, Otx2, or MitF. Additionally, the
RPE cells may show elevated expression levels of alpha integrin subunits
1-6 or 9 as compared to uncultured RPE cells or other RPE cell
preparations. The RPE cells described herein may also show elevated
expression levels of alpha integrin subunits 1, 2, 3, 4, 5, or 9. The RPE
cells described herein may be cultured under conditions that promote the
expression of alpha integrin subunits 1-6. For example, the RPE cells may
be cultured with integrin-activating agents including but not limited to
manganese and the activating monoclonal antibody (mAb) TS2/16. See
Afshari, et al. Brain (2010) 133(2): 448-464. The RPE cells may be plated
on laminin (1 μg/mL) and exposed to Mn2+ (500 μM) for at least
about 8, 12, 24, 36, or 48 hours.

[0118] Table 1 describes characteristics of the RPE cells that may be used
to identify or characterize the RPE cells. In particular, the RPE cells
may exhibit a normal karyotype, express RPE markers, and not express hES
markers. These markers may be used to identify RPE cells in a
heterogeneous population for them to be isolated using the methods
described herein.

[0119] The distinct expression pattern of mRNA and proteins in the RPE
cells of the invention constitutes a set of markers that separate these
RPE cells from cells in the art, such as hES cells, ARPE-19 cells, and
fetal RPE cells. Specifically, these cells are different in that they may
be identified or characterized based on the expression or lack of
expression, which may be assessed by mRNA or protein level, of at least
one marker. For example, the RPE cells may be identified or characterized
based on expression or lack of expression of at least one marker listed
in Table 1. See also Liao, et al. (2010) Human Molecular Genetics 19(21):
4229-38. The RPE cells may also be identified and characterized, as well
as distinguished from other cells, based on their structural properties.
Thus, the RPE cells described herein expressed multiple genes that were
not expressed in hES cells, fetal RPE cells, or ARPE-19 cells. See WO
2009/051671; See also Dunn, et al. (1996) Exp Eye Res. 62(2): 155-169.

[0120] The RPE cells described herein may also be identified and
characterized based on the degree of pigmentation of the cell.
Pigmentation post-differentiation may be not indicative of a change in
the RPE state of the cells (e.g., the cells are still differentiated RPE
cells). Rather, the changes in pigment post-differentiation correspond to
the density at which the RPE cells are cultured and maintained. Mature
RPE cells have increased pigmentation that accumulates after initial
differentiation. For example, the RPE cells described herein may be
mature RPE cells with increased pigmentation in comparison to
differentiated RPE cells. Differentiated RPE cells that are rapidly
dividing are lightly pigmented or unpigmented. However, when cell density
reaches maximal capacity, or when RPE cells are specifically matured, RPE
take on their characteristic phenotypic hexagonal shape and increase
pigmentation level by accumulating melanin and lipofuscin. As such,
initial accumulation of pigmentation serves as an indicator of RPE
differentiation and increased pigmentation associated with cell density
serves as an indicator of RPE maturity. For example, the RPE cells may be
pigmented. For example, the RPE cell may be derived from a human
embryonic stem cell, which cell may be pigmented and expresses at least
one gene that may be not expressed in a cell that may be not a human
retinal pigmented epithelial cell. Further, RPE cells may be derived from
differentiation of embryonic stem cells to produce a heterogeneous
population of embryonic stem cells and RPE cells. The RPE cells may be
morphologically distinguished from the embryonic cells on the basis of
color (e.g., pigmentation), characteristic shape, size, RPE-specific cell
markers, and the absence of ES-specific cell markers. For example, RPE
cells may display a characteristic mottled appearance and cluster to form
dark, pigmented clusters of RPE cells surrounded by undifferentiated,
less pigmented ES cells (e.g., dark clusters of RPE cells surrounded by
translucent ES cells when examined by light microscopy). See FIGS. 1 and
3. The inventors surprisingly discovered that laser microdissection
method may select an area completely within the dark cluster of RPE cells
and thus exclude all contaminating cells of any other type (e.g., ES, ES
cell progeny, other differentiated cells). See FIG. 2. This unexpectedly
allowed for the isolation of a large pure populations of RPE cells
differentiated from ES cells under sterile conditions in a reduced period
of time (compared to manual or chemical selection of RPE cells).
Furthermore, this invention allowed for the isolation of ultra-pure
populations of RPE cells differentiated from ES cells under sterile
conditions in a reduced period of time (e.g., comprising no ES cells
compared to manual or chemical selection of RPE cells).

[0121] Moreover, after the culture containing RPE clusters is treated with
collagenase, the current approach, the desired cells may be difficult to
differentiate because the morphology of clusters in suspension is very
different from their cobblestone appearance (and different for other cell
types that could be of interest), so the operator has to rely on brown
color as primary assessment criteria. However, very dark pigmented cells
may show poor attachment and low survival. Lightly pigmented cells may be
discarded because it is often difficult to differentiate between light
and no pigmentation when using a dissecting microscope (each cluster
would need to be examined individually and from different sides at a high
power microscope which limits its use for cluster harvesting--even if it
could be built into a biosafety hood, such thorough examination would be
time-prohibitive for large scale cell harvest). As a result, unpigmented
clusters may be discarded as well. At the same time, when a culture is
examined prior to harvesting, it has visible large fields where one could
see the cobblestone morphology spreading form dark pigmented to lightly
or non-pigmented areas, and with the laser help those lightly pigmented
cells may also be harvested. Thus, the laser isolation methods described
herein provide a method allowing an operator to identify and isolate less
heavily pigmented RPE cells form a heterogeneous population in an
efficient and rapid manner (as compared to conventional methods).

[0122] Additionally, laser microdissection may be used to isolate RPE
clusters that may contain contaminating cells on the periphery. The
clusters comprising contaminating cells may be isolated using laser
dissection and then allowed to attach to a tissue culture plate. The
clusters may then be further cultured, inspected, and laser dissected a
second time. Further, the methods described herein may be used to
isolated RPE clusters which cannot be easily excised by the laser from
the original monolayer. RPE clusters which may not be cleanly excised by
laser microdissection from the original monolayer may be isolated and
subsequently treated with a collagenase digestion to further purify the
cells (e.g., remove unwanted undifferentiated or other non-RPE cell
types). Again, these RPE cell isolates may be further cultured to allow
for confirmation of their purity and desired phenotype.

[0123] Another aspect of the invention involves the isolation of RPE cells
and other desired cell types from a heterogeneous population of cells
differentiated from ES cells. The laser microdissection methods described
herein may be used when more than one type of cell may be isolated, but
one cell type would be lost if the monolayer was digested (e.g.,
collagenase digestion). For example, in the culture of hES cells en route
towards RPE differentiation, there are, for instance, neural rosettes
which may potentially produce RPE as well as other cell types of the
neural lineage. Using the laser microdissection methods described herein
it may be possible to excise the desired cells without disturbing RPE
clusters and vice versa, remove the RPE cells, and leave the other cell
types (e.g., neural rosettes) to allow for further differentiation.

[0124] Further, the inventors developed a method of isolating cells of
interest based on surface marker expression. Immunostaining requires
either a fluorescence microscope with laser or color reaction. However,
fluorescence may be harmful for the cells, even evaluation of the culture
before the laser is given the coordinates may be damaging, and color
products do not keep the cells alive. To avoid these problems, the
inventors used manual selection of the cells after incubation with
magnetic beads-conjugated antibodies (or the same sandwich indirectly,
antibodies followed by the beads). In particular, DYNAL® beads may be
used and are considerably large compared to beads used in the MACS system
and thus the beads on the cell surface are easily identified. This method
may be used instead of the fluorescent tag for visualization, and after
the selection the beads may not interfere with the cells' growth and may
be removed.

[0125] For example, the heterogeneous cell population may trypsinized to
create a cell suspension. The suspended heterogeneous cell population may
be incubated with magnetic beads-conjugated antibodies and then magnets
used to select the desired cells.

[0126] In contrast with previous preparations, the RPE cells in the
pharmaceutical preparations described herein may survive long term
following transplantation. For example, the RPE cells may survive at
least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. Additionally, the RPE
cells may survive at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks;
at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months; or at least about
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. Further, the RPE cells may
survive throughout the lifespan of the receipt of the transplant.
Additionally, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97,
98, 99, or 100% of the receipts of RPE cells described herein may show
survival of the transplanted RPE cells. Further, the RPE cells described
herein may successfully incorporate into the RPE layer in the
transplantation receipt, forming a semi-continuous line of cells and
retain expression of key RPE molecular markers (e.g., RPE65 and
bestrophin). The RPE cells described herein may also attach to the
Bruch's membrane, forming a stable RPE layer in the transplantation
receipt. Also, the RPE cells described herein are substantially free of
ES cells and the transplantation receipts does not show abnormal growth
or tumor formation at the transplantation site. The methods described
herein resulted in surprisingly ultra-pure isolated populations of RPE
cells differentiated from ES cells under sterile conditions in a reduced
period of time (compared to manual or chemical selection of RPE cells).

[0127] After isolation the cells may remain viable, and may retain the
ability to proliferate (whether in vitro or in vivo). The isolated cells
may be cultured prior to further use, for example to establish larger
populations of cells. Isolated cells may also be used without further
proliferation subsequent to isolation. For example, The RPE cells may be
cultured under conditions to increase the expression of alpha integrin
subunits 1-6 or 9 as compared to uncultured RPE cells or other RPE cell
preparations prior to transplantation. The RPE cells described herein may
be cultured to elevate the expression level of alpha integrin subunits 1,
2, 3, 4, 5, 6, or 9. The RPE cells described herein may be cultured under
conditions that promote the expression of alpha integrin subunits 1-6.
For example, the RPE cells may be cultured with integrin-activating
agents including but not limited to manganese and the activating
monoclonal antibody (mAb) TS2/16. See Afshari, et al. Brain (2010)
133(2): 448-464.

[0128] In another embodiment, the RPE cells may be isolated in accordance
with Good Manufacturing Practice (GMP). In a further embodiment, the RPE
cells may be isolated in accordance with Good Tissue Practice (GTP).

Selection Criteria for Cells

[0129] The method may be used to select and isolate any desired cells. In
one preferred embodiment, the desired cells are cells of a particular
type, such as RPE cells. The cells may be selected (or excluded from
selection) based on any detectable characteristic, including: morphology,
pigmentation, expression of a marker gene, level of expression of a
particular gene, expression of a detectable marker (such as GFP or
another fluorescent protein), autofluorescence (e.g., due to lipofuscin,
elastin, or collagen), viability, surroundings (e.g., colony size,
morphology, local environment) Cells may also be selected (or excluded
from selection) based on any combination of the foregoing types of
characteristics.

[0130] For example, cells exhibiting characteristics of the desired cell
type(s) may be selected for isolation, and optionally cells exhibiting
characteristics of undesired cell type(s) may be excluded from selection.
Selection may be based on any detectable characteristics, including
morphology, pigmentation, detectable markers, and others. For example,
pigmentation may be used for selection (or for exclusion from selection)
of cell types that may naturally contain brown pigmentation in their
cytoplasm: melanocytes, keratinocytes, retinal pigment epithelium (RPE)
and iris pigment epithelium (IPE). Further morphological and other
characteristics may be used to distinguish among these four cell types
before or after isolation. Melanocytes may be distinguished by their
non-epithelial morphology, and keratinocytes may be distinguished because
they do not produce melanin, but rather only take it up via melanosomes.
RPE and IPE cells may be distinguished from melanocytes or keratinocytes
by their typical epithelial cobblestone monolayer appearance. RPE and IPE
may be further distinguished from one another based on molecular,
functional, and morphological characteristics, including: expression of
bestrophin, RPE65, CRALBP, and PEDF by RPE; and behavior of RPE in
culture (little or no pigment may be seen in proliferating RPE cells, but
may be retained in tightly packed epithelial islands or re-expressed in
newly established cobblestone monolayer after the cells became
quiescent). Additional cell types that may be identified based on
pigmentation include neurons of the locus coeruleus (which may contain
neuromelanin granules in their cell bodies that cause light scattering,
resulting in an azure appearance), dopaminergic neurons including neurons
of the substantia nigra (which may contain neuromelanin), pigmented cells
of the brainstem, and pigmented cells of the zona reticularis of the
adrenal gland. Cells may also be identified based on their composition,
e.g., by high numbers of mitochondria (in brown fat). Detection of
mitochondria, golgi, and other structures may be facilitated by contact
with a vital stain, such as those described herein.

[0131] Detectable characteristics of ES cells including but are not
limited to presence in a round colony with clear margins; a high
nucleus/cytoplasm ratio with prominent nucleoli; rounded cells that lie
tightly packed with each other suggesting close cell membrane contact;
and expression of at least one markers characteristic of ES cells such as
OCT-4, Nanog, TRA-1-60, Stage-specific embryonic antigen-3 (SSEA-3),
Stage-specific embryonic antigen-4 (SSEA-4), TRA-1-81, SOX2, and alkaline
phosphatase. Further exemplary markers that may be used to detect ES
cells include at least one of TRA-2-49/6E, growth and differentiation
factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4
(FGF4), embryonic cell-specific gene 1 (ESG1), developmental
pluripotency-associated 2 (DPPA2), DPPA4, telomerase reverse
transcriptase (TERT including hTERT in human cells), SALL4, E-CADHERIN,
Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis, Germ
cell nuclear factor, and Stem cell factor (SCF or c-Kit ligand).
Additionally, desired cells may be distinguished from other cells by
pigmentation. For example, RPE cells are generally darker than other
cells. These characteristics may be used for selection of ES cells or for
their exclusion from selection. For example, cells may be selected from a
population differentiated from ES cells based on presence of detectable
characteristics of a desired cell type, and the absence of at least one
detectable characteristics of ES cells, thereby reducing the risk that
undesired ES cells are among the isolated cells.

[0135] For example, a marker may be detected using a primary antibody may
be directly coupled (e.g., covalently linked) to a detectable label. A
primary antibody may also be indirectly coupled to a detectable label,
which may include coupling via a secondary antibody that binds to a
primary antibody; coupling through binding partners (such as avidin with
biotin, streptavidin with biotin, protein A with Fc, protein G with Fc,
protein A/G with Fc, Protein L with Fc, NeutrAvidin with biotin),
coupling via an antibody binding to an antigen that may be coupled to the
primary antibody, coupling via oligonucleotides (e.g., having
complementary sequences). Additional detectable labels and coupling
methodologies that may adapted for use with the present methods include
those disclosed in U.S. Pat. Nos. 5,281,521; 5,902,727; 5,079,172;
5,665,539; 4,732,847; 6,228,578; 5,132,242; 4,081,245; 4,021,534;
4,481,298; 6,165,798; and 6,117,631. Combinations or chains of the
foregoing coupling methodologies may also be used.

[0136] Exemplary antibodies that may be used with the present methods
include polyclonal, monoclonal, humanized, bispecific, and
heteroconjugate antibodies. For example, polyclonal antibodies may be
raised against whole cells, purified cell surface antigens, or other
preparations as described in Harlow & Lane (1999) Using Antibodies: A
Laboratory Manual and antibodies against the desired cell type(s) may be
depleted, thereby producing polyclonal antibodies that bind other cell
types and allow them to be detected and excluded from selection.

[0137] Cells may also be identified for selection or for exclusion from
selection using staining methods that may be used while retaining cell
viability, such as vital stains. These stains may facilitate
identification of living cells or identification of cells containing or
associated with structures characteristic of a particular cell type.
Exemplary vital stains include eosin (which may be used to stain
cytoplasm, collagen muscle fibers, and other eosinophilic structures),
propidium iodide (a DNA stain that may differentiate necrotic, apoptotic
and viable cells), trypan blue (a diazo dye that is excluded by intact
cell membranes and selectively colors dead cells), erythrosine B
(excluded from live mammalian cells in culture), Hoechst 33258 and
Hoechst 33342 (fluorescent dyes that may label DNA in living cells), and
other Hoechst stains. Additional vital stains include
7-nitrobenz-2-oxa-1,3-diazole-phallacidin (fluorescently stains actin
cytoskeleton in living cells, see Barak, et al. (1980) Proc Natl Acad Sci
USA 77(2): 980-984); liposomes containing
N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine
(C6-NBD-ceramide) (stains Golgi apparatus, see Lipsky & Pagano (1985)
Science 228(4700): 745-7); PicoGreen (stains mitochondrial DNA, see
Ashley, et al. (2005) Exp Cell Res. 303(2): 432-46); phenanthridium
(stains nucleic acids, see U.S. Pat. No. 5,437,980) and other vital
stains known in the art.

[0138] Cell types that may be differentiated from cultured hES cells and
isolated using the presently disclosed methods include, but are not
limited to, ocular cells such as RPE, RPE-like cells, RPE progenitors,
IPE cells, vision-associated neural cells including intemuncial neurons
(e.g. "relay" neurons of the inner nuclear layer) and amacrine cells
(interneurons that interact at the second synaptic level of the
vertically direct pathways consisting of the
photoreceptor-bipolar-ganglion cell chain--they are synaptically active
in the inner plexiform layer and serve to integrate, modulate and
interpose a temporal domain to the visual message presented to the
ganglion cell), retinal cells, lens cells, rods, cones, or corneal cells.
These cells may be identified based on their morphology, pigmentation,
expression of characteristic markers, appearance upon contact with a
stain, expression of a fluorescent protein, and other detectable
characteristics as known in the art and described above.

[0139] The foregoing methods may be used to isolate desired cells while
excluding undesired cells. In a preferred embodiment the undesired cell
will comprise cells which if administered to a subject could cause an
adverse reaction or disease. Specific examples include virally infected
(e.g., HIV, hepatitis) cells, other diseased or aberrant cells (e.g.,
cancerous, precancerous and cancer stem cells), certain immune cells such
as T lymphocytes, and the like which if administered to a recipient,
could result in infection, disease, or other adverse reaction such as an
adverse immune reaction (e.g., GVHD), or result in the proliferation of
undesired cells. Other exemplary undesired cells that may be excluded
include cell types other than the desired cell types, even though such
cells in general may confer little risk of causing adverse reaction or
disease. Undesired cell types may be identified by the presence of a
detectable characteristic, such as morphology and/or expression of a
marker.

[0140] In an exemplary embodiment, cells are excluded from selection if
they exhibit expression of an undesired cell marker. An undesired cell
marker may be specific for one undesired cell type or may indicate
several possible undesired cell types. Any marker (or combination of
markers) may be used so long as it allows desired and undesired cells to
be differentiated. Additionally, an undesired cell marker may exhibit a
frequency of "false positive" binding to the desired cell type. Cells
that express an undesired cell marker may be treated as undesired cells
(e.g., exclusion of that cell from selection and optionally exclusion of
cells within a chosen distance of that cell from selection) even though
that cell may also exhibit a characteristic indicative of a desired cell
type. Combinations of markers may be used to simultaneously indicate a
variety of undesired cell types, e.g., the "Lin" markers intended to
distinguish between hematopoietic stem cells and other blood cell types
(see, e.g., Lagasse, et al. (2000) Nature Medicine 6: 1229-1234). Thus,
exemplary embodiments include detection of multiple undesired cell
markers and treating any cell that detectably expresses any undesired
cell marker as an undesired cell type. Optionally, multiple undesired
cell markers may be detected in a manner that does not distinguish among
them, for example using multiple antibodies directly or indirectly
coupled to the same fluorophore. As one specific example, multiple
undesired cell markers may be detected using primary antibodies sharing a
common binding moiety (e.g., an Fc of a particular species, coupling to
avidin, biotin) and that common binding moiety may be detected using a
fluorophore directly or indirectly coupled to a binding molecule that
recognizes that common binding moiety (e.g., a secondary antibody
specific for that species or another specific binding partner of the
common binding moiety).

[0141] The present invention will now be more fully described with
reference to the following examples, which are illustrative only and
should not be considered as limiting the invention described above.

EXAMPLES

Example 1

Protocol for Laser Microdissection of Living In Vitro Cells

Introduction

[0142] Laser capture microdissection (LCM) is a proven technique for the
isolation of pure cell populations for downstream molecular analysis. The
combined use of UV laser cutting with LCM using an infrared (IR) laser
permits rapid and precise isolation of larger numbers of cells while
maintaining cellular and nucleic acid integrity necessary for downstream
analysis. In this application note, it is shown that these established
techniques can also be used for the isolation of living cells, avoiding
other more laborious methods of cell selection and enabling a wide range
of research applications. This example describes a protocol for the
isolation of living adherent cells and the subsequent recultivation of
homogeneous subpopulations.

Methods

Specimen Preparation

[0143] PEN membrane slide may be hourly rinsed with 100% ethanol and
air-dry prior to use and keep in a sterile environment (e.g., slide
should be completely dry prior to use.) Adherent cells may be trypsinized
from a growth vessel (e.g., plate, flask) using a standard protocol. The
tyrpsin may be deactivated with medium using a standard protocol. About
1-2 mL of trypsinized cells may be resuspended in about 10 mL of fresh
medium. A metal frame membrane slide with chamber may be placed facing up
into a sterile Petri dish. About 1 mL of the cell suspension may be
transferred into the chamber of the frame membrane slide. If necessary,
the slide may be rocked in the Petri dish to completely cover the chamber
area with medium. The lid may be placed on the Petri dish and incubated
using appropriate culturing conditions for the cells until desired cell
confluency is achieved (e.g., replace with fresh medium as needed.

Laser Microdissection Slide Preparation

[0144] The instrument and work area should be thoroughly cleaned,
including pipettors, pipette tip box, with 100% ethanol and RNase
AWAY® or RNaseZap®. A cover glass may be rinsed with 100% ethanol
and air-dry prior to use and in a sterile environment (the cover glass
should be completely dry prior to use.) When cells have reached the
desired confluency, the medium may be removed from the chamber using a
sterile pipette tip. About 950-1,000 μL of fresh medium may be added
to the chamber. A cover glass may be placed over the chamber side of the
frame slide to create a mini-environment for the cell culture, enabling
extended survival and reducing the possibility of the cells drying out.
Care should be taken to reduce the amount of air bubbles formed when
applying the cover glass. A Kimwipe may be used to carefully blot any
excess medium that has seeped outside the cover glass. The slide may be
transported in the Petri dish to a Veritas® or ArcturusXT®
system.

[0145] The slide may be removed from the Petri dish and a Kimwipe soaked
in 100% ethanol may be used to clean the flat side of the frame slide.
The slide should be dried completely. Care should be taken not to rupture
the membrane. The frame slide should be inserted with the chamber and
cover glass facing down (flat side up) onto the Veritas® or
ArcturusXT® instrument and proceed to laser microdissection.

Laser Microdissection Protocol

[0146] CapSure® Macro LCM Caps may be used. Cut and capture may be
performed using light microscopy at 10× or 20×. It is
recommended to identified the desired cell, capture the area, and then
cut with the laser. The visualizer should be turned off (Veritas®
system) and the diffuser should be removed (ArcturusXT® system).

[0147] The cells of interest to be captured may be identified. The Cut
Line feature may be used to draw around cells. The Single Point Capture
feature may be used to apply LCM spots that will fuse LCM membrane to PEN
membrane. It is preferred to apply an adequate number of LCM spots for
the given region.

[0148] A CapSure® Macro LCM Cap may be placed onto the area of the
slide containing cells of interest. LCM laser may be located and fired at
a test LCM shot. If necessary, the laser settings may be adjusted. It is
further recommended that the user confirm that the LCM film makes contact
with PEN film. (The LCM spot will be dark).

[0149] The UV cutting laser may be located. The LCM laser should be
activated first and then the UV cutting laser. The Macro LCM Cap may be
used to a QC station and the presence of cells on the LCM Cap may be
confirmed. The cap may then be moved to an offload station.

[0150] These settings may be used for protocol validation and should be
used as a guideline for the microdissection of live cells. Optimization
of settings may be required, depending on the individual cell
preparation.

Reculturing Captured Live Cells

[0151] The Macro LCM Cap may be removed from the offload station and
inverted. The cap with isolated cells may be placed facing up into a
clean Petri dish. About 50 μL of Hanks' solution may be pipetted onto
the Macro LCM cap film surface. The solution may be pipetted up and down
2-3 times, and the solution disposed. About 50 μL of trypsin-EDTA may
be pipetted directly onto the captured cells on the cap and incubated for
at least about 5 minutes at room temperature. The Petri dish may be
covered with a lid during this incubation. After incubation, trypsin-EDTA
may be pipetted up and down several times to ensure a single-cell
suspension, then transferred the cell suspension into a well of a sterile
chamber slide (or alternate desired growth vessel) containing about 1-2
mL of appropriate cell medium. The chamber slide may be incubated in the
incubator under appropriate conditions. Cell growth may be monitored
using standard culture techniques, changing medium as needed. The
recultured cells may be used as desired for further experiments.

[0153] Human RPE cells were produced by differentiation of human ES cells
essentially as described in U.S. Pat. No. 7,795,025. In brief, hES cell
cultures were maintained and expanded on mouse embryo fibroblast (MEF)
feeder cells, then trypsinized and cultured on low adherent plates
(Costar) until embryoid bodies formed. The embryoid bodies were cultured
until regions containing pigmented cells having epithelial morphology
were formed therein. The embryoid bodies were then digested with enzymes
(trypsin, and/or collagenase, and/or dispase), and pigmented cells were
selectively picked, plated, and cultured. After about two weeks in
culture at low density, the cultured cells lost their pigmentation, but
after another two to three weeks in culture regions of pigmented cells
having a cobblestone, epithelial-like morphology again appeared. This
pigmentation behavior--temporary loss from cells in proliferating
cultures, and restoration in quiescent (non-proliferating) cultures over
time--is a known characteristic of RPE cells and provided initial
confirmation that the culture contained RPE cells. Further confirmation
was obtained by detecting expression of molecular markers characteristic
of RPE cells. The resulting cultures of RPE cells were passaged and
expanded for further use.

Example 3

Isolation of Viable RPE Cells Using Laser Microdissection

[0154] Culture containing RPE cells differentiated from human ES cells
were produced as described in the preceding example. Laser
microdissection was then used to isolate islands of pigmented epithelial
cells for further culture. ES-derived RPE cells were grown in multiwell
culture plates and maintained as quiescent cultures until pigmented
epithelial islands were perceptible (e.g., at least about 7 days). The
multiwell plate was then placed on a microscope fitted with the
STILETTO® laser system (Hamilton Thorne Ltd., Beverly, Mass.) Islands
of pigmented epithelial cells were then visualized, and the provided
control software was used to manually draw a target zone circumscribing
and immediately outside of each pigmented island. Cells in the target
zone were then ablated by laser pulses which were caused to strike the
target zone by computer-controlled movement of the microscope stage.
After ablation of the target zone, each island of pigmented cells was
then physically removed using a micromanipulator and further cultured.

[0155] The laser-isolated RPE cells were grown in culture to confluence
and then maintained as quiescent cultures until pigmented epithelial
islands were established. Compared to control populations of manually
selected pigmented epithelial cells, the cultures of laser-isolated cells
contained non-pigmented or non-epithelial cells as a proportion of the
total number of cells at the similar levels as manually selected
clusters. See FIG. 5.

[0156] The inventors surprisingly discovered that the laser isolation
method was substantially faster than manual colony picking methods (e.g.,
hours versus days). This is a substantial improvement over manual colony
picking methods because it allows for a large number of cells
(>106) to be isolated at near purity in a shorter time. This more
rapid and effective method of isolating RPE cells from an ES cell
population minimizes the time window required to isolate RPE cells and
maximizes the time window the isolated RPE cells are available for
therapeutic use (e.g., 48 hours). Further, the laser microdissection
method allowed the inventors to more rapidly scale up and greatly
increase the number of RPE cells in a shorter period of time with less
lot-to-lot variance.

Example 4

Comparison of Laser-Isolation Methodologies

[0157] As in the preceding example, ES-derived RPE cells were grown in
multiwell culture plates and maintained as quiescent cultures until
pigmented epithelial islands (surrounded by non-pigmented or
non-epithelial cells) were established. The RPE cells were then
laser-isolated as in the preceding example, except that the target zones
were drawn inside the pigmented epithelial islands (instead of
immediately outside of the pigmented epithelial islands). The target
zones were inside of the boundary of each pigmented epithelial island
within 1-2 microns. See, e.g., FIG. 2. The pigmented cells were then
isolated and cultured as in the preceding example.

[0158] Compared to the laser-isolated cells of the preceding example, the
cultures of laser-isolated cells contained a smaller proportion of
non-pigmented or non-epithelial cells. Thus, laser isolation by cutting
within the boundaries of the pigmented epithelial islands produced
higher-purity RPE cultures than laser isolation by cutting just outside
of the boundaries of the pigmented epithelial islands.

Example 5

Multiple Rounds of Purification to Produce Higher Purity RPE Cultures

[0159] RPE cells are produced from hES cells and then laser-purified as
described in the preceding examples (with laser cutting either
immediately surrounding or within pigmented epithelial islands). The
laser-purified RPE cells are cultured until pigmented epithelial islands
appear. A second-round of laser-isolation is then carried out, resulting
in a twice-isolated population of RPE cells. Cultures arising from
twice-isolated cells contain an even greater proportion of pigmented
epithelial cells. The twice-isolated cells may again be cultured until
pigmented epithelial islands appear, and yet again laser isolated to
produce a three times-isolated population of pigmented epithelial cells.
Further rounds of laser isolation may be performed until a desired degree
of purity is achieved.

Example 6

Laser Isolation of Other Eye Cell Types

[0160] A population of cells is differentiated from embryonic stem cells
using the method described in Example 1. A desired eye cell type (such as
ocular cells including RPE, RPE-like cells, RPE progenitors, IPE cells,
vision-associated neural cells, internuncial neurons, amacrine cells,
retinal cells, lens cells, rods, cones, or corneal cells) are identified
based on morphology, pigmentation, expression of characteristic markers,
appearance upon contact with a stain, or other detectable
characteristics. An antibody to a marker characteristic of the desired
cell type (coupled directly or indirectly to a detectable label) may be
used to facilitate detection. Cells of the desired type are then isolated
for further culture. An initial isolation is performed using laser
isolation or other means (e.g., mechanical picking). The isolated cells
are then cultured. The desired cell type may then undergo at least one
rounds of laser isolation, thereby producing a more pure culture of the
desired cell type. The isolated cells may then be used for cell-based
therapy in a human or non-human animal.

[0161] While the invention has been described by way of examples and
preferred embodiments, it is understood that the words which have been
used herein are words of description, rather than words of limitation.
Changes may be made, within the purview of the appended claims, without
departing from the scope and spirit of the invention in its broader
aspects. Although the invention has been described herein with reference
to particular means, materials, and embodiments, it is understood that
the invention is not limited to the particulars disclosed. The invention
extends to all equivalent structures, means, and uses which are within
the scope of the appended claims.

[0162] Although the invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it was
obvious that certain changes and modifications may be practiced within
the scope of the appended claims. Modifications of the above-described
modes for carrying out the invention that are obvious to persons of skill
in cell biology, molecular biology, and/or related fields are intended to
be within the scope of the following claims.

[0163] All publications (e.g., Non-Patent Literature), patents, patent
application publications, and patent applications mentioned in this
specification are indicative of the level of skill of those skilled in
the art to which this invention pertains. All such publications (e.g.,
Non-Patent Literature), patents, patent application publications, and
patent applications are herein incorporated by reference to the same
extent as if each individual publication, patent, patent application
publication, or patent application was specifically and individually
indicated to be incorporated by reference.